Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein as though set forth in full.
Recombinant factors VII, VIIa, VIII, IX, and protein C are important pharmaceuticals useful in the treatment of traumatic bleeding complications (6), hemophilia (7), and sepsis (8). These proteins play important roles in the coagulation cascade (39), which comprises plasma and membrane proteins that are responsible for arrest of blood loss at sites of injury.
Factors VII, IX, and protein C belong to a family of vitamin K-dependent proteins that are modified post-translationally to contain γ-carboxyglutamic acid (Gla), Ca++ binding amino acid residues (2). The modification is carried out by the vitamin K-dependent γ-carboxylation system located in the endoplasmic reticulum (ER) (2). Two essential enzymes of the system are 1) the vitamin K-dependent γ-carboxylase, an integral membrane protein of 92 kDA which requires reduced vitamin K (vit.K1H2) as cofactor and 2) the warfarin sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR), which produces the cofactor (2).
A major problem with production of recombinant vitamin K-dependent coagulation factors for use as pharmaceuticals has been poor recovery of functional proteins from the cell medium (9). It has been shown that poor recovery results from, 1) incomplete γ-carboxylation of the secreted proteins (9) and 2) incomplete removal of the propeptide by the protease PACE/furin in the Golgi apparatus. Incomplete γ-carboxylation is a significant problem as <10% of the secreted recombinant vitamin K-dependent proteins have been reported to be fully γ-carboxylated and functional (9). It is believed that incomplete γ-carboxylation occurs when an excess of newly synthesized precursors of vitamin K-dependent proteins appear in the ER and exceed the capacity of the cell's γ-carboxylation system to fully modify all of the precursors (9).
Clearly, a need exists for improved methods for producing high levels of the coagulation factors discussed above. It is an object of the invention to provide such methods.